The immediate cause of Huntington's disease is a triplet expansion in the DNA leading to an inherited gene for the protein huntingtin (htt) with an equivalent expansion of a polyglutamine (polyQ) repeat. How this mutated protein sequence triggers the molecular and cellular events leading to HD pathology continues to be debated. There is now general agreement that the large htt containing inclusions observed in HD brains on autopsy, and in cellular and animal models of HD, are probably not the toxic species. However, no evidence as been presented against the possible involvement of smaller aggregates not visible in fluorescence microscopy. This is significant since the only widely accepted known consequence of polyQ expansion to the behavior of polyQ disease proteins like htt is an enhancement of protein aggregation. A number of screening assays for small molecules capable of blocking aggregation have been conducted leading to compounds that block aggregation and suppress toxicity in animal models. One such compound, riluzole, failed to induce a response in human clinical trials, but then it also failed in a animal model preclinical trial, perhaps because of insufficient concentration in the brain. Recently our group has elucidated a new aggregation mechanism for the exon1 fragment of huntingtin containing the polyQ sequence, which depends on a triggering protein unfolding event in the exon1 N-terminus just before the polyQ. Characterization of exon1 aggregation in vitro led to the discovery that there are two aggregation pathways competing for exon1 molecules, each of which produces different aggregation intermediates and/or products. Using a new staining method specific for amyloid-like aggregates of polyQ, we identified a new aggregate in mammalian cells producing exon1 that have been thought to only produce large inclusions of exon1. In this application we propose to develop new high throughput screening assays for identifying aggregation inhibitors in large libraries of small molecules. The premise of this proposal is that we do not know which of the known huntingtin exon1 aggregated species is the most toxic and most likely to contribute to HD. We propose two new screening assays based on our preliminary data. One focuses on finding molecules that will prevent, in vitro, the protein misfolding event in the exon1 N-terminus that we believe triggers a portion of htt exon1 aggregation. The other focuses on preventing the formation in mammalian cell culture of the amyloid-like aggregates of exon1 that are different from the large inclusions previously focused on in screening and molecular mechanism studies. We propose to develop these assays and fine-tune them to a state ready for high throughput screening. We will also develop a variety of secondary screening assays that will be required to eliminate false positives from future high throughput screens. We believe that these assays could lead to the discovery of new classes of inhibitors capable of slowing the progression of HD. PUBLIC HEALTH RELEVANCE: This research project is immediately relevant to human health in that it proposes to develop new screening assays for drug discovery of potential Huntington's disease therapeutics. If successful, these assays could very quickly be consigned to high throughput screens to identify new candidate molecules.